title: Preparation and assay of C-glucosyltransferase from roots of Pueraria lobata.
abstract: C-glucosyltransferase (EC 2.4.1.X) is one of the key enzymes for the biosynthesis of puerarin. This paper describes the methodology in purification and assay of the enzyme for the first time in Puerarin lobata (Wild.) Ohwi. C-glucosyltransferase from roots of P. lobata was extracted and partially purified by (NH4)2SO4 saturation. The effects of pH, temperature, and substrate concentration on the activity of the enzyme were investigated. The properties of the puerarin produced by C-glucosyltransferase were studied by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The peak activity of C-glucosyltransferase was detected in fraction of by 80% saturation of (NH4)2SO4 and the optimal conditions for enzymatic reaction were 35.5 micromol l(-1) of isoliquiritigenin and 560 micromol l(-1) of UDP-G at pH 8.1, 28 degrees C for 1 h. Mn2+ at 1 mmol l(-1) and Al3+ at 1 mmol l(-1) increased the enzyme activity, while Mg2+ inhibited its activity. The enzyme activity in Nicotiana tabacum and P. lobata were detected under the above assay conditions. Higher activity was found in roots than in leaves and stems of P. lobata, while no enzyme activity was detected in leaves of N. tabacum. It was the first time that activity of C-glucosyltransferase, which transforms isoliquiritigenin to puerarin, was detected in P. lobata.
authors.affiliation: Guangdong Key Lab of Biotechnology for Plant Development, College of Life Sciences, South China Normal University, Guangzhou - 510 631, China.
authors.forename: Gang
authors.initials: G
authors.lastname: Chen
authors.forename: Xuan
authors.initials: X
authors.lastname: Wu
authors.forename: Wen-Ling
authors.initials: WL
authors.lastname: Zhou
authors.forename: Ling
authors.initials: L
authors.lastname: Li
mesh_terms: D000645:Ammonium Sulfate; D005964:Glucosyltransferases / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D018517:Plant Roots / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D029908:Pueraria / Q000737:chemistry*; D013696:Temperature
chemical_list: D005964:Glucosyltransferases; D000645:Ammonium Sulfate